PROJECT SUMMARY Mitochondrial DNA (mtDNA) sequence variations have been causally implicated in cardiometabolic diseases (CMDs). However, the mechanisms linking mtDNA mutations to CMDs are not fully elucidated. Each human cell contains as many as 10,000 copies of mtDNA. As a result, there are two types of mtDNA mutations ? homoplasmic and heteroplasmic mutations. The latter represent the coexistence of two (or more) mtDNA alleles in the same cell or across cells. For most heteroplasmic mtDNA mutations, the proportion of mutant alleles is low and can only be detected by deep sequencing of mtDNA. Whole genome sequencing (WGS) through NHLBI?s Trans-Omics for Precision Medicine program has generated deep WGS data, including mtDNA, in tens of thousands of individuals, offering an unprecedented opportunity to investigate heteroplasmic mtDNA mutations in relation to CMDs. We propose to develop rigorous methods and a software suite to facilitate association studies of heteroplasmic mtDNA mutations in large samples. The novel methods and the software suite will be open source and available to the broad scientific community to study mtDNA sequence variation with respect to age-related diseases, including CMDs. The body of knowledge generated by these future findings will facilitate the development of new modalities for the diagnosis, prevention, and treatment of age-related diseases.